Abstract Bioorthogonal reactions are chemical reactions that occur in biological systems between two exogenous functional groups in the presence of the naturally occurring reactive groups. These reactions have found extensive applications in basic research and are becoming prominent in medicine, particularly in medical imaging and targeted drug therapies. A bottleneck in the advancement of this technology is the chemistry of the bioorthogonal reaction. All except one of the widely used bioorthogonal reactions, the tetrazine ligation, have a crucial deficiency: slow reaction kinetics under physiological conditions. To overcome the kinetics, large excess of one of the components is used to speed up the reactions, which must then be removed by a purification step. We have uncovered a new bioorthogonal reaction that also breaks the kinetic barrier and can quickly and efficiently perform biomolecular couplings using low concentrations of reagents in aqueous solution at physiological pH. The resulting product is a highly unusual boron-containing heterocycle. The premise of this proposal is that a new bioorthogonal reaction that is complementary and orthogonal to the existing reactions will be an important addition to the repertoire of bioconjugation resources. The goal of this research is to characterize the scope of this new bioorthogonal reaction and its potential applicability for efficient, biocompatible coupling reactions. We propose to accomplish these goals through the following specific aims. (1) We will delineate a structure-reactivity-stability profile of this conjugation reaction through selection and synthesis of structural variants of the reactive pairs, structural analysis of the reaction products under biologically relevant conditions and kinetic analysis of the reactions of these molecules. We will test our hypothesis that this reaction is well suited for protein labeling applications and experimentally determine its orthogonality to and compatibility with widely used bioorthogonal reactions. These data will enable rational selection of a reactive pair to create a conjugate with properties commensurate with the chosen application. (2) We will apply this technology to create anti-cancer antibody-drug conjugates with fast preparation time and efficient use of the cytotoxic agent and assess the specificity and cytotoxicity of these new conjugates. Successful completion of these aims will yield a robust ensemble of reagents that will meet a pressing need in fundamental research and in the development of targeted therapeutics and theranostic agents.